Maternal mRNA Clearance Prevents Precocious Transcription and Genome Instability in Mouse Early Embryos [ATAC-seq]

Oocytes accumulate abundant maternal mRNAs during the growth stage of meiotic maturation after transcriptional silencing, and early embryo development prior to zygotic genome activation (ZGA). During the oocyte-to-zygote transition, the selective and stepwise clearance of maternal transcripts is a prerequisite for the initiation of zygotic development. Maternal mutations in genes involved in mRNA clearance, including Pabpn1l, Cnot6l, and Btg4, lead to early embryonic arrest at the one-to-two cell stage in both mice and humans; however, the direct underlying mechanism is unclear. In the present study, optimized low-input sequencing methods of newly transcribed RNAs (neo-RNAs) were used, and the results provide evidence that the arrested zygotes abnormally accumulate transcription-associated DNA damages and R-loops in pronuclei. Ectopic R-loop formation and DNA strand breaks are accompanied by increased chromatin accessibility and leakage of maternal genes that should be turned off following fertilization. Abnormal R-loop accumulation causes the retardation of DNA replication and S-phase arrest during the first mitotic cell cycle. Overexpression of RNase H1 in maternal Pabpn1l knockout zygotes can remove genomic R-loops and partially reverse developmental defects. Collectively, the conjoint analysis of chromatin accessibility and neo-RNA transcription indicated that the failure of maternal mRNA clearance in mouse zygotes results in leakage transcription and extra R-loop formation, which is the primary reason for DNA replication stress, genome instability, and zygotic cell cycle arrest. Overall design: Each sample contained 50 zygotes collected from mated female mice 28 h after the hCG injection. After removing zona pellucida with acidic M2 (pH 2.5), zygotes were washed with DNase/RNase-free 0.2% BSA and collected in DNA-lowering binding tubes. The lysis buffer was added directly after sample collection. Tn5 enzyme in Chromatin Profile Kit for Illumina (Novoprotein; N248) was used to fragment chromatin at 37 ? on Thermo Mixer (TS100) with 1000 rpm for 30 min and stopped using stop buffer. After extraction with DNA extract beads, DNA fragments were amplified for 15 cycles using the following PCR conditions: 72 ? for 3 min; 98 ? for 30 s; and thermocycling at 98 ? for 15 s, 60 ? for 15 s and 72 ? for 8 s; following by 72 ? 2 min. After the PCR, libraries were purified with 1.2 × DNA clean beads, followed by fragment selection with 0.5×/0.7× beads.